1. Metropolis Health Services
Hemoglobin Electrophoresis
Dr Nisha S Ahmad
Chief of Lab Services
Metropolis Health Services

2. Agenda Brief overview of hemoglobin The globin genes The Thalassemias
Structural hemoglobinopathies
Testing

3. Hemoglobin
4 Heme groups
4 polypeptide chains

4. Hemoglobin structure A B B A
heme

5. Hemoglobins in normal adults
HbA
HbF
HbA2
95-98%
~1%
<3.5%

6. The Globin Genes

8. Hemoglobin Type
Name
Components
Adult A
22 A2
22
Fetal F
22
Embryonic
Portland
22
Gower 1
22
Gower 2
22
Abnormal H 4
Bart’s 4

9. Hemoglobin Types Quantity Alpha Thal >95% HbH HbA2 (α2 δ2) <3.5%
HbA (α2 β2)
>95%
HbH
HbA2 (α2 δ2)
<3.5%  -
HbF (α2 γ2)
<2.0%
Hb Barts

10. Hemoglobinopathy
An inherited mutation of the globin genes leading to a quantitative or qualitative abnormality of globin synthesis

11. Thalassemia - Defined
A family of genetic anemias characterized by a reduced rate of production of 1 or more globin subunits of hemoglobin (Hb)
Symptoms are caused by the deleterious effects of the normally produced subunits that are now in excess

12. Pathophysiology
Excess alpha chains precipitate and form inclusion bodies that associate with the RBC cell membrane
Cause membrane damage and shortened cell survival
Large scale destruction of precursor cells in bone marrow
Decreased B production causes increased δ production and an elevated A2 (α2δ2)

13. Types of B-globin mutations
B0 – No B-globin chains are produced
B+ - some beta chains produced Decreased

14. Alpha thalassemia AA / AA Normal AA / A -
Mild microcytosis (Silent Carrier)
AA / - -
A - / A -
Mild microcytosis (Trait or Carrier)
(cis vs trans)
A - / - -
Hemoglobin H disease- clinically variable
- - / - -
Hydrops Fetalis (Alpha Thal Major)

15. α- and β-thalassemia Alpha Thalassemia
Deletions of alpha- globin gene (s)
Symptoms can begin in fetal life
Complicated inheritance – 4 alpha genes
Beta Thalassemia
Nonsense, splice and frameshift mutations in beta-globin gene
Symptoms begin in infancy/childhood
Simple AR inheritance; genotype-phenotype correlation

16. Structural variant - Defined
Abnormal globin protein that is produced at a normal rate, with varying consequences
Oxygen affinity, stability and function

17. Normal

18. Normal Thalassemia

19. Normal Structural Variant

20. Laboratory Investigation
CBC-MCV,MCH,RDW
Tests
Hemoglobin electrophoresis
Cellulose acetate: Alkaline pH
Citrate agar: Acid pH
Capillary Electrophoresis
HPLC
IEF
DNA

21. Preanalytical
EDTA sample
Age
H/O Transfusion
Area of Residence E/D/C

22. SCREENING ANTENATAL PROFILE HbA1c ANEMIA PROFILE PRE MARITAL
NEONATAL PROFILE

23. Manual System

24. Cellulose Acetate
pH-8.6
In a alkaline solution ,Hb molecules have a net negative charge and move towards the anode

25. Cellulose Acetate Hb Electrophoresis
- A F A +
C/E/O S/D/G
NORMAL
BTT

26. Cellulose Acetate Hb Electrophoresis
- A2/C S F A+
Normal
Hb AS
Hb SS

27. Cellulose Acetate Hb Electrophoresis
- A2/C/E S /D F A+
Normal
Hb AS
HB AD

28. Citrate Agar Electrophoresis
Citrate agar electrophoresis at an acid Ph provides ready separation of hemoglobins that migrate together on cellulose acetate
S from D and G
C from E and O

29. Citrate Agar Hb Electrophoresis
C S A F _
Normal

30. Citrate Agar Hb Electrophoresis
C S A F _
Normal
Sickle trait

31. HPLC
Separation based on interaction between Stationary Phase & Mobile Phase
Stationary Phase is Analytical Cartridge; Mobile Phase is Buffer
Compounds are separated to target analytes according to physical properties:
- size, shape, charge, hydrophobicity & affinity for other molecules
Bound analytes elute off the stationary phase by manipulating the mobile phase

32. Contd…
Charged particles (matrix) bind reversibly to sample molecules (proteins, etc.)
Desorption is then brought about by increasing the salt concentration or by altering the pH of the mobile phase

33. Automated system;precalibrated column and gradient
HPLC
Automated system;precalibrated column and gradient

34. Stationary Phase: Cation Exchange Cartridge
Direction of flow
Detector
Carboxyl groups attached to a resin base

35. Hemoglobin Introduction
Positively charged hemoglobin fragments in the hemolysate attach to the carboxyl groups at varying strengths.

36. Starting Gradient: Low Ionic Strength Buffer
The gradient starts with a low % of Buffer B (high % Buffer A)
At this gradient, hemoglobin fragments with an ionic strength lower than the buffer gradient, such as A and F, are displaced from the cartridge and pass into the detector

37. Ending Gradient: High Ionic Strength Buffer
As the % of the High Ionic Strength Buffer B increases, the more hemoglobin fragments will be displaced
Once the gradient is 100% Buffer B all remaining hemoglobin fragments, including any variant hemoglobins such as S, D and C, will be removed

38. CHROMATOGRAMS
Peak
Output
Time
Area RT

40. Normal HPLC Graph
HbA F A2

41. Hb C
Hb H
HbF
HbS
Hb J
Hb Köln
HbE
Hb O
HbD
Hb Q

42. A2 A2 Suggests <3.5% Normal 3.5-8.0% BTT /(Megaloblastic anemia)
11-15%
Hb Lepore
20-40%
HbE
30-48%
HbD

44. Hb Lepore

45. HbE

46. HbD

47. Hb EE/DD

48. HbF F Suggests <2% Normal <5% HPFH/Pregnancy/ Aplastic anemia
2-15%
HPFH
BTT
Sickle
10-15%
Delta Beta Thalassemia
>50%
Beta Thal Major
HPFH (homozygous)
Delta Beta Thalassemia (homo)

49. Hb F
1.Delta beta Thal Trait
2.HPFH+IDA
1.Iron Studies
2.DNA Analysis

50. HPLC GRAPH OF CHILD Hb F= 100% Hb A2= 0% HbA0 =0% CBC: Hb = 6.8 g/dl
MCV = 67.3 Fl
MCH = 21.3 pg
RDW = 24.7%

51. HPLC GRAPH MOTHER Hb F = 7.0% HbA2 = 2.6% HbA0 = 90.4% CBC: Hb = 11.9
MCV = 64.7
MCH = 21.1
Impression:
S/o Delta beta thal trait

52. HPLC GRAPH FATHER Impression S/O Delta beta thal trait HbF = 8.0%
HbA2= 2.8%
HbA0 = 89.2%
CBC: Hb = 13.4
MCV = 68.8
MCH = 22.4
Impression
S/O Delta beta thal trait

53. HbF

54. Low A2 Iron Deficiency Anemia Alpha Thalassemia trait HbH Disease
Delta Thalassemia
Delta –Beta Thalassemia

55. Peak before 1 min
Sample Integrity
Icteric sample
HbH

56. HbH
Age-8 yrs
Hb-7.8g/dl
MCV-61 fl
MCH-17.8pg

57. HbH

58. Extended Retic stain
HbH inclusions

59. Capillary Electrophoresis
Advantages
HbH
HbE vs HbD
Disadvantages
Preanalyticals (Cap piercing model)
Interpretation

60. Limitations…. Silent carriers Alpha Thalassemia
DNA analysis-gold standard

61. DNA analysis Silent carriers Prenatal period 5 common mutations

62. Conclusion….. Clinical Suspicion CBC PBS Reticulocyte count
Specific tests
Electrophoresis
HPLC
Capillary Electrophoresis
DNA analysis-Pre natal/gold standard

63. Questions ?